What are the common problems associated with western blot analysis?
One of the most common problems in western blot analysis occurs when SDS-polyacrylamide gels are overloaded with protein sample. This results in protein bands, which appear smeared or aggregated, particularly in the absence of DTT in the loading sample buffer. Usually, 1-10 µl of induced culture should be loaded. If cell pellet from 1 liter culture is resuspended in 100 ml column buffer, 1-2 µl of each sample should be loaded. We also load 1:10 and 1:100 dilutions of the cell extract. TCEP [tris-(2-carboxyethyl)phosphine] (PIERCE) can be used as a reducing agent in SDS-PAGE Sample Buffer in place of DTT to improve the quality of SDS-PAGE. Unlike DTT, TCEP should not cause cleavage of the fusion protein. Alternatively, samples may be prepared with DTT-containing SDS-PAGE Sample Buffer as long as a control sample (induced cell extract) prepared with a DTT-free sample buffer is included for comparison during western blot analysis or stained SDS-PAGE. This will prevent one from overestimat
